Cryopreserved tissues are defined as tissues which have been frozen in the presence of one or more cryoprotective agents. Cryoprotective agents are defined as compounds which are used to reduce damage to cryopreserved tissues/cells during the freezing, storage, and/or thawing processes associated with cryopreservation.
A variety of tissues are preserved by cryopreservation in the presence of cryoprotective agents. Methods and protocols for the cryopreservation of human heart valves have been extensively described in the scientific literature since the early 1970's, however the method described in U.S. Pat. No. 4,890,457 is representative of these early methods. Of concern to the present invention is the removal of cryoprotective agents from such tissues following or simultaneous with thawing just prior to transplantation into a patient. Most cryoprotective agents used to protect the cryopreserved tissue during cryopreservation are harmful to tissues surrounding the implant due to time and temperature dependent chemical and physical damage and it is generally deemed important to remove these agents from the thawed tissue prior to transplantation. Cryoprotectant removal techniques that have been described include, procedures where the concentration of cryoprotectant is reduced via a timed series of additions of lower osmolality solutions, and procedures where removal is accomplished by adding the tissues to a given volume of lower osmolality solution such as described in U.S. Pat. No. 5,160,313. The approach using a series of additions of solutions to remove cryoprotectant from tissues is time consuming and requires particular attention to details such as elution time, temperature, and composition of the diluting solution. The approach using a given volume requires less attention to details such as elution time, temperature, and composition of diluting solution, however the osmotic shock to the cellular population is rapid and can lead to dramatic cell swelling and subsequent cell damage and/or death (osmotic shock). This osmotic shock may be mitigated by including impermeant solutes in the diluting solution, however, such diluting solutions typically result in a tissue containing an osmotic pressure of between 400 to 800 mOsm which is dramatically greater than the approximate 290 mOsm associated with normal tissue. The present inventive method solves the problem associated with prior art methods by providing a user-friendly, continuous-multi-step dilutional process for removing cryoprotective agents from cryopreserved tissue while maintaining the resultant osmotic pressure within an acceptable normal range. Using a wash-out solution of 280-290 mOsm/kg water results in greater osmotic shock but yields "final" tissue which is iso-osmolar. Using wash-out solution of 500-600 mOsm/kg water lessens the initial osmotic shock but results in "final" tissue which is hyperosmolar.